GSM6619659: Embryo, E15.5, replicate 1, spatial ATAC; Mus musculus; ATAC-seq
Crosslinked and permeabilized sections were tagmented d according to OMNI ATAC-seq 3 at 37°C for 1 h under gentle shaking (300 rpm every 5 minutes) using 2 μl Tn5 in tagmentation mix (25 μl 2x TD buffer, 16,5 μl DPBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20). To stop the tagmentation and strip the transposase from DNA, sections were incubated with 50 mM EDTA while ramping down to 30°C for 10 minutes. To hybridise the tagments to the barcoded surface oligonucleotides, we then incubated the sections with a 2 μM solution of splint oligonucleotide (in 3X SSC buffer containing 0.01% Triton-X100, 0.8 μg/μl Proteinase K and 2.5% PEG8000) overnight at 30°C. Next, the sections were rinsed in 2X NEB 2.1 buffer and subsequently incubated with ligation and polymerization solution (1X NEB2.1 containing 3U T4 DNA polymerase, 2000U T4 DNA ligase, 100 μM dNTPs, 1 mM ATP, all from NEB) and incubated at 18°C for 4h. Tissue removal was then performed using 2 mg/ml Proteinase K in PKD-buffer (Qiagen), and incubated at 56°C for 30 minutes (shaking at 300 rpm). The slides were then sequentially washed in 2X SSC 0.1% SDS, 0.2X SSC and 0.1X SSC and finally spin dried. Spatially barcoded ssDNA fragments were released from the surface by denaturation with 0.08N KOH at room temperature for 10 minutes and then quenched in 10 μl of 1M Tris pH7. The denatured fragments were pH adjusted with sodium acetate and cleaned with MinElute Reaction Cleanup Kit (Qiagen, 28204). The eluted DNA was then amplified using PCR using Partial.R1 and Ad2.short oligonucleotides for 15 cycles using PrimeSTAR Max DNA Polymerase mix (Takara, R045B). The amplified products were purified using 0.9X SPRI beads and i7-indexed in a second PCR (4 cycles) using PE1.0 and Ad2.X (where X is the sample index from 4) oligonucleotides and KAPA HiFi HotStart Mix (Roche, KK2602). The final indexed libraries were cleaned up using 0.8X SPRI beads and adjusted to the desired molarity based on the concentrations measured using Qubit HS dsDNA Assay Kit (Thermo, Q32854) and the average fragment size from HS DNA Bioanalyzer kit (Agilent, 5067-4626). Pooled libraries were then sequenced on Illumina Nextseq 550 or 2000 instrument using custom sequencing oligonucleotides for Read1 and Index2 (CustomR1 and CustomI2). We sequenced 65 bases for reads 1 and 2 (genomic sequence), 28 bases for i5 (spatial barcode and UMI), and 8 bases for i7 (sample index).